Molecular Ecology Resources (2008) 8, 23–34 doi: 10.1111/j.1471-8286.2007.01963.

DNA BARCODING
Blackwell Publishing Ltd

A ribosomal DNA-based framework for the detection and

quantification of stress-sensitive nematode families in
terrestrial habitats
M A RT I J N H O LT E R M A N ,* K ATA R Z Y N A RY B A R C Z Y K ,* S V E N VA N D E N E L S E N ,* H A N N Y VA N
M E G E N ,* PA U L M O O Y M A N ,* R E Y E S P E Ñ A S A N T I A G O ,† T O M B O N G E R S , J A A P B A K K E R * and
JOHANNES HELDER*
*Laboratory of Nematology, Graduate School for Experimental Plant Sciences, Wageningen University, Binnenhaven 5, 6709 PD
Wageningen, The Netherlands, †Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus
‘Las Lagunillas’ s/n, Edificio B3, 23071 Jaén, Spain

Abstract
Indigenous communities of soil-resident nematodes have a high potential for soil health
assessment as nematodes are diverse, abundant, trophically heterogeneous and easily
extractable from soil. The conserved morphology of nematodes is the main operational
reason for their under-exploitation as soil health indicators, and a user-friendly biosensor
system should preferably be based on nonmorphological traits. More than 80% of the most
environmental stress-sensitive nematode families belong to the orders Mononchida and
Dorylaimida. The phylogenetic resolution offered by full-length small subunit ribosomal
DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding
several discrepancies between morphology and SSU rDNA-based systematics, Mononchida
families (indicated here as M1–M5) are relatively well-supported and, consequently, family-
specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longi-
doridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more
variable large subunit rDNA (≈ 1000 bp from the 5′-end) was sequenced for 72 Dorylaimida
species. Sequence analysis revealed a subclade division among Dorylaimida (here defined
as D1–D9, PP1–PP3) that shows only distant similarity with ‘classical’ Dorylaimid system-
atics. Most subclades were trophically homogeneous, and — in most cases — specific
morphological characteristics could be pinpointed that support the proposed division. To
illustrate the practicability of the proposed molecular framework, we designed primers
for the detection of individual subclades within the order Mononchida in a complex DNA
background (viz. in terrestrial or freshwater nematode communities) and tested them in
quantitative assays (real-time polymerase chain reaction). Our results constitute proof-of-
principle for the concept of DNA sequence signatures-based monitoring of stress sensitive
nematode families in environmental samples.
Keywords: LSU ribosomal DNA, nematode, RT-PCR, soil community analysis, SSU ribosomal DNA

Received 2 July 2007; revision accepted 1 August 2007

in water (films) in densities up to several millions of indi-

Introduction
Nematodes are among the most widespread and abundant
invertebrates in soils and (freshwater and marine) sedi-
ments. These relatively small, vermiform organisms reside
Correspondence: Johannes Helder, Fax: + 31 317 484254; E-mail:
Hans.Helder@wur.nl
viduals and up to 100 species per square metre (Anderson
2001). The high density and the variety of trophic ecologies
represented within this phylum — nematodes may feed on
bacteria, fungi, algae, other nematodes, plants (in)verte-
brates or a combination of these (Yeates et al. 1993) — render
them a key position in the soil food web (De Ruiter et al.
1998; Bongers & Ferris 1999). Furthermore, nematodes

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Journal compilation © 2007 Blackwell Publishing Ltd
24 D N A B A R C O D I N G
themselves serve as a food source for a wide range of soil
inhabitants (Bongers & Ferris 1999). Nematodes show a
broad range of sensitivities towards disturbances such as
subtle temperature changes, changing moisture conditions,
exposure to pollutants, and changes in the nutritional
status of their environment. Taking into consideration
that nematodes — unlike bacteria and fungi — can easily
be extracted from soil, they have a great potential as
indicators for soil health (recently reviewed by Dmowska
& Ilieva-Makulec 2004).
In terrestrial habitats, the great majority (> 80%) of the
environmental stress-sensitive nematode families — as
indicated by the c-p values 4 and 5 (c, colonizer; p, persister;
Bongers 1999) — belongs to two orders, namely the
Dorylaimida and the Mononchida (16 and six families,
respectively). Despite their common overall sensitivity to
environmental stresses, their responsiveness towards
different kinds of physical, chemical or biological distur-
bances is diverse. Therefore, the monitoring of shifts in
Dorylaimida and Mononchida communities in terrestrial
and freshwater habitats at family or even genus level is
ecologically relevant (Ettema & Bongers 1993; Bongers
1999; Bongers et al. 2001; Georgieva et al. 2002). As com-
pared to Mononchida, Dorylaimida are highly speciose;
Jairajpuri & Ahmad (1992) estimated that more than 10% of
all currently known nematode species belong to this order.
The range of trophic ecologies represented by these two
orders is just marginally smaller than the diversity within
the phylum Nematoda as a whole. Its members can be
found in all soil types as well as in freshwater environ-
ments, while remarkably they are absent in marine
habitats. In comparison to the well-known free-living
nematode Caenorhabditis elegans (1 mm in length), Dory-
laimida and Mononchida are relatively large (typically
1–5 mm), have long generation times (months instead of
3–4 days for C. elegans), and produce a relatively low
number of eggs. The low reproduction rates imply that
Dorylaimida and Mononchida populations will only
slowly recover from disturbances. The high sensitivity to
pollution could be partially explained by the permeability
of their cuticle. Generally spoken, these nematodes have
relatively permeable cuticles (Hollis 1961; Premachandran
et al. 1988). All these characteristics combined make
members of the Dorylaimida and Mononchida sensitive
indicators of the impact of environmental stress on soil life.
Dorylaimida display a mosaic of morphological charac-
ters and are notoriously difficult to identify, even for experts.
As it is unclear which characters are relevant for the estab-
lishment of phylogenetic relationships within this order
and which characters suffer from homoplasy, there have
been numerous rearrangements of genera, families and
superfamilies within this order (for a historical overview
see Jairajpuri & Ahmad 1992). Apart from the scarcity of
informative morphological characteristics, the potential
of Mononchida and Dorylaimida as sensitive bio-indicators
is underexploited because routine analysis of soil samples
is very time-consuming (a single mass-slide takes on
average 2 h), and because only adults are taken into
consideration (juveniles cannot always be identified).
It is concluded that a nematode-based biosensor should
be based on nonmorphological traits.
Molecular analysis has become a powerful tool to clarify
evolutionary relationships, and generally spoken, well-
resolved relationships are a strong basis for DNA sequence
signature-based taxon identification. The small subunit
ribosomal DNA (SSU rDNA) gene has proven to be useful
for the reconstruction of phylogenetic relationships among
nematode taxa (Aleshin et al. 1998; Blaxter et al. 1998; Rusin
et al. 2003; Holterman et al. 2006), and recently, a sub-
division of the phylum into 12 clades has been proposed
(Holterman et al. 2006). Members of the orders Dorylaim-
ida and Mononchida were shown to reside within a single
major clade (Clade 2), and the representatives were distrib-
uted over two well-resolved order-specific branches. In
contrast to the families within the order Mononchida, the
phylogenetic relationships within the family-rich order
Dorylaimida were fully unclear (Holterman et al. 2006).
The low diversity of the SSU rDNA within the order
Dorylaimida prompted us to sequence a part of the
more variable large subunit (LSU) ribosomal DNA gene
(≈ 1000 bp from the 5′-end). In many cases (45), both the SSU
(full length) and LSU (fragment) rDNA were sequenced
from the same individual nematode. In this study, we
present SSU and LSU rDNA-based phylogenetic analyses
of Mononchida and Dorylaimida, and show the possibilit-
ies of using SSU and LSU rDNA-based sequence signatures
for the quantitative detection of individual stress-sensitive
nematode families in environmental samples.
Materials and methods
Taxon sampling
Nematodes were collected from various habitats through-
out the Netherlands, and extracted from the soil using
standard techniques. Prior to DNA extraction, individual
nematodes were identified using a light microscope (Zeiss
Axioscope) equipped with DIC optics. A CCD camera
(CoolSnap, RS Photometrics) was used to take a series of
digital images from each nematode.
SSU rDNA sequences
Part of the SSU rDNA sequences used in this study came
from an earlier work on the phylogeny of the Nematoda
(Holterman et al. 2006), and new sequences were acquired
as described in this study. SSU rDNA sequences were
deposited at GenBank under Accession nos EF207244
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd

to EF207254. For a full list of sequences used for this
study, see Tables S1 and S2, Supplementary material.
DNA extraction, LSU rDNA amplification and
sequencing
Single nematodes were transferred to a 0.2 mL polymerase
chain reaction (PCR) tube containing 25 μL sterile water.
An equal volume of lysis buffer containing 0.2 m NaCl,
0.2 m Tris-HCl (pH 8.0), 1% (v/v) β-mercaptoethanol and
800 μg/mL proteinase K was added. Lysis took place in a
Thermomixer (Eppendorf) at 65 °C and 750 r.p.m. for 2 h
followed by 5 min incubation at 100 °C. Lysate was used
immediately or stored at –20 °C. LSU rDNA was ampli-
fied using either primer 28–61for or 28–81for (28–61for,
5′-GTCGTGATTACCCGCTGAACTTA-3′; 28–81for, 5′-TTA-
AGCATATCATTTAGCGGAGGAA-3′) in combination with
either primer 28–1006rev or 28–1032rev (28–1006rev,
5′-GTTCGATTAGTCTTTCGCCCCT-3′; 28–1032rev, 5′-TC-
GGAAGGAACCAGCTACTA-3′). PCR was performed in a
25-μL final volume containing 3 μL of 100× diluted crude
DNA extract, 0.1 μm of each PCR primer and a ‘Ready-To-Go
PCR bead’ (Amersham). The following PCR protocol was
used: 94 °C, 5 min; 5× (94 °C, 30 s; 45 °C, 30 s; 72 °C, 70 s)
followed by 35× (94 °C, 30 s; 54 °C, 30 s; 72 °C, 70 s) and
72 °C, 5 min. Gel-purified (Marligen) amplification products
were cloned into a TOPO TA vector (Invitrogen) and sequenced
using standard procedures. Newly generated LSU rDNA
sequences were deposited at GenBank under the following
Accession numbers: AY592994–AY593065 and EF207234–
EF20743.
Sequence alignment
SSU rDNA sequences were supplemented with pub-
licly available sequences (Table S1) and aligned using the
clustal w algorithm as implemented in bioedit 5.0.9
(Hall 1999) and manually improved using secondary struct-
ure information from arthropods [http://www.psb.ugent.be/
rRNA/secmodel/index.html, in accordance with Wuyts
et al. (2000)].
Newly generated nematode LSU rDNA sequences were
supplemented with one publicly available sequence
(Xiphinema rivesi AY210845). The outgroup consisted
of Mononchida, namely: Mononchus tunbridgensis,
Mononchus truncatus and Anatonchus tridentatus. The
LSU rDNA sequences were aligned using the same methods
as for the SSU rDNA sequences, and further improved
with secondary structure information from yeast (http://
www.psb.ugent.be/rRNA/varmaps/Scer_lsu.html; see
also Ben Ali et al. (1999)). The final alignment consisted
of 74 partial LSU rDNA sequences (each sequence spans
about 1000 bp from the 5′-end onwards) and contained
1309 aligned positions (including gaps).
© 2007 The Authors
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D N A B A R C O D I N G 25
Phylogenetic analyses
The SSU rDNA trees were constructed using Bayesian
inference. modeltest selected the general time reversible
(GTR) model with invariable sites and gamma distribution
as the best fitting models for both SSU data sets. In essence
the data set was analysed as described in Holterman et al.
(2006), except for now the gamma parameter was included.
The Dorylaimia SSU rDNA data set was run for 2 000 000
generations and the burn-in was 100 000 generations (Fig. 1).
The Dorylaimida SSU rDNA data set was run for 1 800 000
generations and the burn-in was 500 000 generations
(Fig. 2).
Three different methods were used to construct a phylo-
genetic tree from the LSU rDNA; neighbour-joining (NJ),
maximum parsimony (MP) and Bayesian inference (BI).
modeltest 3.06 (Posada & Crandall 1998) was used to
determine the most appropriate nucleotide substitution
model. Both the likelihood ratio test and the Akaike infor-
mation criterion selected the GTR model with invariable
sites (Ι) and a γ-shaped distribution of substitution rates
(Γ) as the best fitting substitution model. The neighbour-
joining tree was constructed using paup* 4.0b10 (Swofford
1998) using the model parameters calculated by model-
test. The resulting tree was bootstrapped 1000 times. The
MP tree was also constructed using paup*, the default
parameters were used. This tree was bootstrapped 1000
times as well. The Bayesian tree was constructed using the
program mrbayes 3.1.2 (Ronquist & Huelsenbeck 2003).
The alignment was divided into a stem and a loop parti-
tion according to the secondary structure. For both parti-
tions, GTR + Ι + Γ was used. The stem regions were
analysed under the Doublet model. It is noted that the
Doublet + GTR model explained our data only marginally
better that the GTR only approach (data not shown). The
default flat priors were used for the parameters and the
parameters were unlinked between the partitions. Four
independent runs with different random starting trees
were performed. Each run was made with four Markov
chains and run for 3 million generations with a sample
frequency of 200 generations. The first 200 000 genera-
tions were discarded as burn-in. The sampled trees
from the four runs were combined in a single 50%
majority-rule tree. The program tracer version 1.2.1
(Rambaut & Drummond 2005) was used to check if all para-
meters had converged. The program macclade version 4.0
(Maddison & Maddison 2000) was used to infer the
ancestral character states of several traits along the Bayesian
LSU rDNA tree.
Detection and quantification of individual subclades
To detect and quantify clusters as defined in Fig. 1 (M1–
M5), subclade-specific SSU rDNA-based primers were
26 D N A B A R C O D I N G

Fig. 1 SSU rDNA-based Bayesian phylogeny of the subclass Dorylaimia (Clade 2 in Holterman et al. 2006). Numbers near nodes represent
posterior probabilities. The coloured bar indicates to which order, family, and subclade (M1–M5) a species belongs. The colour of the species
name indicates the feeding type (according to Yeates et al. 1993). A ‘G’ behind the species name indicates the sequence was acquired from
GenBank.

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Journal compilation © 2007 Blackwell Publishing Ltd
 D N A B A R C O D I N G 27

Fig. 2 SSU rDNA-based Bayesian phylogeny of the order Dorylaimida. Numbers near nodes represent posterior probabilities. The coloured
bars indicate to which suborder, family and subclade a species belongs. Only species names belonging to well-resolved subclades are given
(for detailed phylogenetic tree see Fig. S1). A ‘G’ behind the species name indicates the sequence was acquired from GenBank.

designed using the software package arb (Ludwig et al.
2004). An alignment of about 1200 full-length SSU rDNA
sequences covering a substantial part of the nematode
biodiversity in terrestrial and freshwater habitats was used
as input file to identify subclade-specific sequence motives.
Potential close nontargets were selected by changing the
arb mismatch setting to — at most — four nucleotides.
One or two mismatches were always considered as close
nontargets unless they were positioned very close to the
3′-end of the foreseen PCR primer. Three and four mis-
matches were only included if they were clustered and
positioned in the 5′-end region.

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28 D N A B A R C O D I N G

Table 1 Primer combinations used for the detection and quantification of Mononchida subclades as defined in Fig. 1. Real-time PCR results
are presented in Fig. 4. Close nontarget species belonging to the subclass Dorylaimia are given in italics

Annealing
Subclade Primer combination temperature
identifier (F, forward; R, reverse) Close nontargets (°C)

M1 F: 5′-CGATCCGTCGGTGTTAAATAT*T Prionchulus punctatus 1 63

R: 5′-CTCG*AGCTGATGACTCGA*A Mononchus truncatus 1
Haliplectus sp. 1
Prismatolaimus dolichurus 2
Pratylenchus pratensis
M2 F: 5′-CGCATTTATTAGACCAAAACCAG* Unidentified mermithid species 64
R: 5′-TAGAAGACCCAGTTAAACTCCTT* Mesocriconema xenoplax 1
Malenchus andrassyi 1
Ditylenchus dipsaci 9
M3 F: 5′-CGAGCTTCTTAGAGGGACAG* Mylonchulus rotundicaudatus 2** 65
R: 5′-CCAATTCTTACCAGAAAAGGTTTTAA Opisthodorylaimus sylphoides**
Granonchulus sp. 1
Diphtherophora obesa 1
Trischistoma sp. 1**
Eumonhystera filiformis 1**
Steinernema glaseri1**
Prochromadora sp. 1**
M4 F: 5′-CGATCCGTCGGTGTTAAG* Mylonchulus sp. 1 62
R: 5′-CCAATTCTTACCAGAAAAGGTTTTAA Mylonchulus sigmaturus 3
Mylonchulus brachyuris 2
Mylonchulus rotundicaudatus 2
Mononchus truncatus 1
Clarkus papillatus 1
Coomansus parvus 1
Granonchulus sp. 1
Bathyodontus mirus 1
Cryptonchus tristis 1
Prionchulus punctatus 2
Prismatolaimus dolichurus 2
Euteratocephalus sp. 1
M5 F: 5′-GACGAAGAATTTTATATGTTTTTTGTG* Anatonchus tridentatus 1 63
R: 5′-GGTTGTAAAGCACACTGCTATTC* Granonchulus sp. 1
Coomansus parvus 1

*LNA; **starts giving a positive signal in later cycles.

Subclades are defined in Fig. 1, and for each of the sub-
clades, the closest nontargets are shown in Table 1. Primer
combinations as presented in Table 1 were tested using
cloned SSU rDNA fragments. Bacterial clones harbouring
a TOPO TA vector with an SSU rDNA fragment of interest
were grown at 37 °C in 2 mL of LB medium supplemented
with 100 μg/mL of ampicillin. Plasmid extraction was
performed using the Wizard Plus Minipreps DNA Purifica-
tion System (Promega). DNA concentrations were meas-
ured with a NanoDrop spectrophotometer and adjusted
to 10 ng/μL. For Q-PCR application 3 μL of 1000× diluted
template was mixed with a subclade-specific primers
(end concentrations for both primers 200 nm) and 12.5 μL
iQ SYBR Green Supermix (Bio-Rad) in a total reaction
volume of 25 μL. In order to increase the specificity
occasionally locked nucleid acids (LNAs) were incorporated
(Table 1). Thermal cycling was performed on a Bio-Rad
MyiQ thermal cycler (Bio-Rad) and consisted of 98 °C
for 3 min; followed by 60 cycles of 98 °C for 30 s, subclade-
specific annealing temperature (Table 1) for 1 min and 72 °C
for 30 s.
Results and discussion
Currently, nematode community analysis for ecological
soil classification invariably includes time-consuming light
microscopy-based identification (mostly till family level)
and counting of relatively small samples (typically 75 up

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Journal compilation © 2007 Blackwell Publishing Ltd

to 200 individuals). In most cases, nematode families are
defined on the basis of a series of morphological characters
that are not always visible in juvenile life stages. For a
transformation of prevalent nematode community analysis
tools into DNA sequence signature-based methods, it is
relevant to know whether DNA sequence data do or do not
confirm the existence of these nematode families as they
are currently defined. Here, this is investigated for members
of the subclass Dorylaimia, with a focus on Mononchida
and Dorylaimida; two orders whose members live exclusi-
vely in terrestrial and freshwater habitats and share a
high sensitivity to environmental stress. Subsequently,
we show how subclade-specific DNA sequence sig-
natures can be used for life-stage independent, large-
scale analysis of terrestrial and freshwater nematode
communities.
Phylogeny of the subclass Dorylaimia
Six orders of the Dorylaimia are represented in our analysis
(Figs 1 and 2): Dorylaimida, Mononchida, Mermithida
(insect parasites), Trichinellida (animal parasites), Diocto-
phymatida (animal parasites) and Isolaimida (an order that
comprises only a single family with one genus; Isolaimium).
SSU rDNA sequence data offer a remarkably good resolution
within the Mononchida, Mermithida and Trichinellida
(Fig. 1), whereas the phylogenetic resolution among repre-
sentatives of the Dorylaimida was poor (Fig. 2). Addition
of sequences from representatives of the Cryptonchidae
and the Soboliphymatidae — both positioned basally in
two major branches (see Fig. 1) — resulted in intraclade
relationships that differ from the relationships presented
by Mullin et al. (2005) and Holterman et al. (2006). The
current data set suggests a basal node that defines the
Dorylaimida on the one hand, and the Mononchida,
Mermithida, Trichinellida, and Dioctophymatida on the
other. Within the second group, a sister relationship is
observed between the Mononchida and Mermithida,
and the Trichinellida and Dioctophymatida. As animal
parasites are not taken into consideration in ecological soil
assessment, the withinorder relationships of Mermithida,
Trichinellida, and Dioctophymatida will not be discussed
here. The order Isolaimida was placed outside the Dorylai-
mia, a confirmation of a result that was recently published
by Mullin et al. (2005). We will focus on the Mononchida
and Dorylaimida as the numerous representatives of these
orders are stress-sensitive as reflected by their high c-p
values (4 or 5 on a 1–5 scale as defined at family level
(Bongers 1990).
Mononchida — phylogenetic relationships
SSU rDNA-based phylogenetic relationships among
Mononchida are to some extent similar to the current
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Journal compilation © 2007 Blackwell Publishing Ltd
D N A B A R C O D I N G 29
classification of the order. Most striking is the positioning
of the Bathyodontidae and the Cryptonchidae, two families
harbouring exclusively bacterial feeding nematodes
(Yeates et al. 1993), at the base of the Mononchida subclade.
This would suggest that predatory (see below) Mononchida
and insect-parasitic Mermithida arose from bacterivorous
ancestors. In this respect, it is noteworthy that the food
preference of nematodes may change during their life
cycle; contrary to adults (and J3/J4), initial juvenile stages
of several members of the Mononchida feed on bacteria
(e.g. Yeates 1987). The remarkable and firm placement of
the Mermithidae (insect parasites) within the Mono-
nchida confirms previous findings by Blaxter et al. (1998),
Mullin et al. (2005) and Holterman et al. (2006). Apparently,
there are no morphological characters that support this
positioning.
The family Mylonchulidae was shown to be polyphyletic
as Granonchulus did not cluster with representatives of the
genus Mylonchulus (M1 in Fig. 1). The characters consid-
ered to be diagnostic for the Mylonchulidae are the strong
tapering of the stoma (mouth-like opening) at its base, and
the arrangement of the denticles in transverse rows (Zullini
& Peneva 2006). However, the stoma of Granonchulus is
ovoid, not tapering strongly at the base, and members of
this genus have only one transverse row of denticles
(instead of multiple); the remaining denticles are ordered
in longitudinal rows. Hence, morphological data are avail-
able that seem to support our SSU rDNA-based results.
The Mononchidae also turned out to be paraphyletic,
with Mononchus being a sister group of Mylonchulus, separ-
ate from Coomansus, Clarkus and Prionchulus (M2 in Fig. 1).
There is morphological support for this separation. Both
Mononchus and Mylonchulus have well-developed tail glands
and a (reduced) spinneret (a cuticular cone connected to
the caudal glands). On the contrary, Coomansus, Clarkus
and Prionchulus (M3 in Fig. 1) have only rudimentary tail
glands and no spinneret (Zullini & Peneva 2006). In addi-
tion, Mononchus has a transverse rib on each ventrosub-
lateral wall of the stoma and Mylonchulus has denticles on
the ventrosublateral walls arranged in transverse rows. In
contrast, Coomansus, Clarkus and Prionchulus have longitu-
dinal ridges on their ventrosublateral stoma walls (Zullini
& Peneva 2006). These characteristics support the results
from our phylogenetic analysis.
The Anatonchidae appeared to be paraphyletic, too.
Representatives of the genus Anatonchus (M4 in Fig. 1)
are placed at the base of a subclade that includes most
Mononchidae and Mylonchulidae. Our analysis suggests
that it is probably not possible to define Anatonchidae-
specific DNA sequence signatures that would cover all
members of the genera Anatonchus and Miconchus.
Bathyodontidae (M5 in Fig. 1) are represented
by two species only. So far, this family seems to be
monophyletic.
30 D N A B A R C O D I N G
Dorylaimida — SSU rDNA-based phylogenetic
relationships
Within the Dorylaimida, two suborders are distinguished,
the Dorylaimina and the Nygolaimina (De Ley et al. 2006).
These are characterized by the nature of their stoma
(Coomans 1964). The Dorylaimina are equipped with an
axial odontostyle, whereas the Nygolaimina have a mural
tooth. In the SSU rDNA-derived tree (Fig. 2), the Nygolaimina
are placed in a single, well-supported subclade (D9). SSU
rDNA sequence data do not allow for the deduction of
family relationships among Dorylaimina. The only family
for which all members are present in a single well-supported
cluster is the plant-parasitic family Longidoridae. However,
because it is part of a large polytomy, the relationship
between Longidoridae and other Dorylaimida families
could not be established.
Dorylaimida — LSU rDNA-based phylogenetic
relationships
The conserved nature of the SSU rDNA among represen-
tatives of the Dorylaimida prompted us to sequence a part
of the LSU rDNA in addition to the SSU rDNA (≈ 1000 bp
from 5′ end). In many cases (45 of 72 sequences), both SSU
and LSU rDNA were amplified from the same individual
nematode. The LSU rDNA-based phylogram is constructed
on the basis of 75 sequences: 72 representatives of the Dory-
laimida and three members of the Mononchida. The LSU
trees show a better resolution within the Dorylaimida,
although a large basal polytomy still remains. The Bayesian
tree (Fig. 3), parsimony tree (Fig. S2, Supplementary material)
and neighbour-joining tree (Fig. S3, Supplementary material)
are nearly identical, and therefore, only the Bayesian tree is
depicted here. All parameters of the evolutionary model
for the Bayesian tree had converged after a burn-in of
200 000 generations.
As there is limited congruence between the current
family subdivision of the Dorylaimina and the subclades
suggested by LSU rDNA sequence data, it was decided to
define 12 subclades, D1–D9 and PP1–PP3, that are well-
supported by molecular data. Dorylaimina systematics has
been subject to numerous revisions, and we investigated
whether morphological support could be found for the
currently proposed subclade division:
D1. Being quite heterogeneous from a morphological point
of view, D1 shows several general (although not totally
common) patterns (Fig. S4, Supplementary material). (i) It
groups several long-tailed taxa which currently are dispersed
in separate families (and even superfamilies): Dorylaimidae
(Dorylaimoidea), Mydonomidae (Tylencholaimoidea) and
Belondiridae (Belondiroidea). (ii) It groups several taxa
(Paractinolaimus, Dorylaimus, Opisthodorylaimus and Meso-
dorylaimus) which display sexual dimorphism in the tail
shape (females have an elongated or cone-shaped tail and
males a rounded tail), a feature not found elsewhere in the
studied taxa. (iii) It assembles several taxa which share the
feature of being opisthodelphic (= uterus directed posteriorly)
in total or in part (Opisthodorylaimus, Ecumenicus, Dorylai-
moides, Oxydirus), an infrequent feature in Dorylaimidae
and its relatives; remaining subclades are dominated by
didelphic (= two uteri) or predominantly didelphic taxa,
with the exception of PP2 (Pungentus species) and D5
(Tylencholaimus species).
D2. This cluster consists of Aporcelaimellus and Allodorylaimus
species that belong to the Aporcelaimidae and Qudsiane-
matidae, respectively. To the best of our knowledge, there
are no morphological characters supporting this clade.
D3. This cluster includes several Qudsianematidae (Epidory-
laimus, Eudorylaimus and Thonus), Enchodelus (Nordiidae–
Pungentinae) and Prodorylaimus (Dorylaimidae) and it was
also distinguished on the basis of SSU rDNA data (Fig. 2).
Although these taxa share several characters, none of these
characters are unique for this subclade (Fig. S4). These
shared characteristics are: (i) guiding ring simple (double
in Enchodelus and Prodorylaimus), (ii) pharynx widening
near or slightly behind the middle, (iii) vagina sclerotized,
and (iv) tail shape equal in both sexes. Although none of
these characteristics is D3-specific by itself, their combina-
tion is fairly unique. The only other genus which combines
all these four characteristics is Allodorylaimus, which is placed
in clade D2.
PP1. This subclade consists of Microdorylaimus, Longidorella
and Eudorylaimus cf. minutus. This subclade is charac-
terized by having a pharynx that spans about a third of
their total body length (possibly related to their relatively
small size — 0.4–0.7 mm).
PP2. It consists solely of the genus Pungentus.
D4. This consists of members of the Qudsianematidae
subfamilies Carcharolaiminae and Discolaiminae. These
subfamilies were previously regarded as a separate family
— the Discolaimidae (Siddiqi 1969; see also De Ley et al.
2005) — and based on these results it seems reasonable to
reinstate this family.
D5–D9, PP3. D5, PP3 and D6 are monophyletic subclades
that so far appear to correspond with the families Tylen-
cholaimidae, Longidoridae and Tylencholaimellidae,
respectively. D7 includes representatives of the genus
Sectonema, the only genus within the subfamily Sectonema-
tinae. Hence, this cluster corresponds to a subfamily as
currently defined within the Qudsianematidae. D8, defined
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
 D N A B A R C O D I N G 31

Fig. 3 LSU rDNA-based Bayesian phylogeny of the order Dorylaimida. Numbers near nodes represent posterior probabilities. The coloured
bar indicates to which (sub)family a species belongs. The Dorylaimida are divided into 12 subclades (D1–D9, PP1–PP3). The colour of the
species name indicates the feeding type (according to Yeates et al. 1993).

© 2007 The Authors

Journal compilation © 2007 Blackwell Publishing Ltd
32 D N A B A R C O D I N G
here by a single LSU rDNA sequence only, corresponds to
the Qudsianematidae subfamily Chrysonematinae. D9
covers Nygolaimidae (Nygolaimina) and this subclade
could be clearly distinguished on the basis of SSU rDNA
sequence data as well (Fig. 2).
The origin of plant parasitism within the order
Dorylaimida
Plant parasitism arose at least three times independently
during the evolution of the phylum Nematoda, one time
within the order Dorylaimida (Longidoridae), and two times
in the (infra)orders Triplonchida and Tylenchomorpha
(Blaxter et al. 1998). For the latter case, Holterman et al.
(2006) provided molecular support for a long-standing
hypothesis stating that plant parasitic nematodes arose
from fungivorous ancestors (Maggenti 1971). We inves-
tigated the positioning of the Longidoridae (PP3) vis-à-vis
the two fungivorous subclades D5 and D6. However,
the current data set provided insufficient resolution to
make a statement about the origin of the Longidoridae.
Remarkably, two more groups of plant feeders within
the order Dorylaimida, members of the genera Longidorella
and Pungentus (ectoparasites of higher plants, Yeates et al.
1993; but also see Trudgill 1976), evolved independently
from the Longidoridae. From our LSU rDNA data, we con-
clude that Longidorella (subclade PP1) presumably arose
from an omnivorous ancestor. The current LSU rDNA tree
provides no insight into the possible feeding type of the
ancestor of Pungentus.
The use of DNA sequence signatures for quantitative
detection of Mononchida subclades
Unique DNA sequence signatures (unique among 1200
full length SSU rDNA sequences from all over the phylum
Nematoda) were determined for the five subclades (M1–
M5) within the order Mononchida as defined in Fig. 1. On
the basis of these signatures, primers were designed that
would work under similar annealing temperatures, and
these were tested for their specificity (Fig. 4). It is noted
that close nontargets as given in Table 1 do not necessarily
belong to the Dorylaimia. On the contrary, except for M5,
all primer combinations tested were shown to have close
nontargets that are phylogenetically completely unrelated
to the target sequence. In four cases (M1, M2, M4 and M5),
primers were shown to be highly specific as hardly any
signal could be detected even after 60 PCR cycles. Primers
designed for M3 were slightly less specific, but the ΔCT (CT
— cycle number at which the fluorescent signal exceeds a
given threshold value) was still around 20. It is concluded
that nematode subclades as defined here provide a firm
basis for the development of assays for the detection and
quantification of stress sensitive nematode families in soils.
DNA sequence signature-based identification of
Dorylaimida subclades
LSU rDNA sequence analysis resulted in a subdivision of
the Dorylaimida into nine free-living subclades (D1–D9),
and three clusters that include multiple parasites of higher
plants (PP1–PP3). Contrary to LSU, SSU rDNA data are
available from a considerable number of taxa well spread
over the phylum Nematode. Hence, subclades are preferably
defined on the basis of specific, shared SSU rDNA sequence
motives. As can be seen in Fig. 2, this is achievable only for
D3, D9, and PP1–PP3. The remaining subclades will be
defined by shared LSU rDNA motives. Currently, the LSU
rDNA data base is dominated by representatives of the
Dorylaimida and the Tylenchomorpha, and consequently,
subclade-specific primers could potentially have cross
reactivity outside Clades 2 and 12. It should be noted that
— as compared to SSU rDNA — the relatively high degree
of variability of the LSU rDNA genes among nematodes
reduces the chances of unwanted cross reactivity consi-
derably. Nevertheless, we are planning to alleviate possible
uncertainties about specificity by the development of SSU-
rDNA-based suborder Dorylaimina-specific primers. In the
absence of (major) cross reactivity, the total Dorylaimina
signal minus the D3, D9, and PP1–PP3 signals should be
similar to the D1–D2 plus D4–D8 signals.
Further development of this detection system will
include the determination of the average quantitative PCR
signal yield per family or subclade. For this, we are
currently generating series of 1, 5, 10, 50 and 100 micro-
scopically determined individuals from single genera. By
determining the PCR signal yield per genus, we will get
insight in the within-family variation. As nematodes with
individual families or subclades tend to have similar body
sizes (e.g. Bongers 1994), we expect a moderate variation,
and this would enable us to define factors that translate
the quantitative PCR signal into a reasonable estimate
of the number of individuals to which this signal is cor-
responding. Finally, parallel investigations of field samples
on the basis of morphological and molecular characteristics
will be needed to further validate this entirely novel
approach for the analyses of soil and freshwater nematode
communities.
Conclusion
Although the potential of nematodes as indicators for the
ecological condition of soil and freshwater sediments is
widely recognized (e.g. Bongers & Ferris 1999), the large-
scale exploitation of this group so far has been hampered
mainly by their conserved morphology. The molecular
framework for the detection of two major, trophically hetero-
genous groups of stress-sensitive nematodes combined
with the relatively simple quantitative PCR-based analysis
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd

tool as presented here offers great perspectives for the
exploitation of this group as it lifts — at least in part — the
need for specialist taxonomic expertise, detects all develop-
mental stages (instead of — mainly — adults), facilitates the
analysis of relatively large and numerous soil and/or
sediment samples, and greatly reduces the sample-
handling time.
Acknowledgements
This work was supported by the Dutch Technology Foundation
(STW) grant WBI4725. Further support was received from Senter-
Novem, an agency of the Dutch Ministry of Economic Affairs
(IS043076).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
D N A B A R C O D I N G 33
Fig. 4 Real-time PCR amplification curves showing the detection
of trophically homogeneous subclades within the order
Mononchida as defined in Fig. 1 on the basis of full length SSU
rDNA sequences (M1–M5). Target species, closest nontargets,
primers and annealing temperatures are given in Table 1.
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Supplementary material
The following supplementary material is available for this article:
Table S1 GenBank Accession numbers of SSU sequences.
Table S2 GenBank Accession numbers of LSU sequences.
Fig. S1 Complete Bayesian SSU tree Dorylaimida
Fig. S2 Maximum parsimony LSU tree
Fig. S3 Neighbour-joining LSU tree
Fig. S4 Character tracing Dorylaimida LSU tree
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